Methods for control of flux in metabolic pathways through enzyme relocation

a metabolic pathway and enzyme technology, applied in the field of metabolic pathway flux control through enzyme relocation, can solve the problems of limiting overall yield, affecting the production of compound, etc., and achieves the effect of high level production

Active Publication Date: 2011-11-10
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

During the cell growth phase, the relocated enzyme, which may be sequestered in the periplasm, for example, does not affect the pathway flux. In order to initiate the production phase

Problems solved by technology

Yet embedding a novel biochemical pathway in the metabolic network of a host cell or modifying the expression of enzymes in a native biochemical pathway can disrupt the subtle regulatory mechanisms that the cell has evolved over millennia.
Indeed, the final yield of a compound is often limited by deleterious effects on the engineered cell's metabolism that are difficult to predict due to limited understanding of the complex interactions that occur within the cell.
The unregulated consumption of cellular resourc

Method used

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  • Methods for control of flux in metabolic pathways through enzyme relocation
  • Methods for control of flux in metabolic pathways through enzyme relocation
  • Methods for control of flux in metabolic pathways through enzyme relocation

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of Shikimic Acid

Shikimic acid is an intermediate in the chorismate biosynthetic pathway, where the key entry enzyme is 2-dehydro-3-deoxyphosphoheptonate aldolase (3-deoxy-D-arabino-heptulosonate-7-phosphate, DAHP, synthase). DAHP synthase catalyzes the first committed step in shikimate production by converting the central metabolites phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) to DAHP. In E. coli, there are three DAHP synthase enzymes—AroG, AroE, and AroF—encoded by genes aroG, aroE, and aroF, respectively. It is common that feedback resistant versions of these enzymes (Kikuchi et al., Appl. Environ. Microbiol. (1997) 63:761; Ray et al., J. Bacteriol. (1988) 170:5500; Weaver and Herrmann, J. Bacteriol. (1990) 172:6581) are used to ensure maximal activity.

In this example, a DAHP synthase gene is modified to contain various periplasmic signal sequences (periplasmic leader peptides) targeting the enzyme to the periplasm. Expression optimization, evaluation of v...

example 2

Growth Effects and Activity of Periplasmically-Expressed AroG

Periplasmic expression of DAHP synthase. A library of plasmids is constructed containing the gene coding for AroG (Genbank Acc. no. AAC73841.1) modified with various periplasmic signal sequences targeting the enzyme to the periplasm. The DNA sequences of primers used to construct the coding sequences for the periplasmic leaders tested are set forth in Tables 4 and 5. DNA sequences coding for a set of periplasmic signal sequences are added to the aroG gene through PCR amplification using the following primers:

TABLE 4Primers Used to Add Periplasmic Targeting Signals to aroGLeaderPrimerSequencenoneF 5′gcaattcggtctcccatgaattatcagaacgacgatttacgcatc(SEQ ID NO: 11)R 3′gaattcgcggccgcttacccgcgacgcgcttttac(SEQ ID NO: 12)OmpAF 5′gcaattcggtctcccatgaaaaaaacggcaattgcgatagcg(SEQ ID NO: 13)R 3′gaattcgcggccgcttacccgcgacgcgcttttac(SEQ ID NO: 14)StIIF 5′gcaattcggtctcccatgaaaaaaaatattgctttcctgctcg(SEQ ID NO: 15)R 3′gaattcgcggccgcttacccgcgacgc...

example 3

Cell Free Production of Isobutanol and / or 1-butanol

Current methods for production of isobutanol in E. coli rely on over-expression of the E. coli enzymes of valine biosynthesis IlvI,H,C,D in concert with overexpression of two heterologous enzymes: the alcohol dehydrogenase 2 enzyme of S. cerevisiae (ADH2, GenBank AAA34411.1) and the 2-keto-acid decarboxylase enzyme of L. lactis (KivD, GenBank CAG34226.1) (see, e.g., Atsumi et al., Nature (2008) 451:86). Similarly, production of 1-butanol requires the overexpression of the same two heterologous enzymes (ADH, KivD) combined with overexpression of IlvA and LeuABCD enzymes of isoleucine and leucine biosynthesis in E. coli (Atsumi ibid). Accumulation of higher alcohols (e.g., isobutanol, 1-butanol, n-butanol) is toxic at very low levels, 2% (w / v) (Atsumi ibid; Reyes et al., Plos One (2011) 6:e17678) resulting in poor cell growth and poor product titers when these pathways are active in growing E. coli.

Periplasmic relocation of the key e...

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Abstract

Genetically manipulated cells, lysates of such cells, systems, and methods of use thereof are provided, where one or more enzymes in a pathway of interest are genetically modified to incorporate a peptide sequence that provides for relocation of the protein, e.g., to the periplasm, so as to sequester the enzyme, and where the enzyme controls flux in the pathway of interest.

Description

BACKGROUND OF THE INVENTIONProduction of chemicals via synthetic enzymatic pathways in microbial hosts has proven useful for many important classes of molecules, including isoprenoids, polyketides, nonribosomal peptides, bioplastics, and chemical building blocks. Due to the inherent modularity of biological information, synthetic biology holds great potential for expanding this list of microbially produced compounds even further. Yet embedding a novel biochemical pathway in the metabolic network of a host cell or modifying the expression of enzymes in a native biochemical pathway can disrupt the subtle regulatory mechanisms that the cell has evolved over millennia. Indeed, the final yield of a compound is often limited by deleterious effects on the engineered cell's metabolism that are difficult to predict due to limited understanding of the complex interactions that occur within the cell. The unregulated consumption of cellular resources, metabolic burden of heterologous protein pr...

Claims

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Application Information

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IPC IPC(8): C12P1/00C12N9/00C12N1/21C12N1/19C12N5/10C12N1/15C12N1/11C12N15/63C12N1/13
CPCC07K2319/034C12N9/1029C12N9/88C12N15/625Y02E50/10C12P7/42C12P7/625C12P23/00C12P7/16C12N1/00C12N1/06C12N15/52C12N15/70C12P5/007
Inventor SWARTZ, JAMES R.
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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